Abstract
P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by omega-conotoxin MVIIC. 125I-omega-Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by omega-conotoxins GVIA or MVIIA (Ki > 1 microM), which are selective for N-type calcium channels. Solubilized 125I-omega-conotoxin MVIIC receptors were specifically recognized by antibodies directed against alpha1A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20-40%) of omega-conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained alpha1A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q-type calcium channels, which may optimize their location within domains of calcium influx.
Highlights
P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by -conotoxin MVIIC. 125I--Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by -conotoxins GVIA or MVIIA (Ki > 1 M), which are selective for N-type calcium channels
-Conotoxins provide useful probes for the biochemical assay of voltage-dependent calcium channels. GVIA and -conotoxin MVIIA (MVIIA) are selective antagonists of N-type channels in which the transmembrane pore is constituted by an ␣1B subunit
In contrast MVIIC, which is highly homologous to MVIIA, blocks P, Q, and N-type calcium channels in neurons (Hillyard et al, 1992; Randall and Tsien, 1995), and inhibits currents induced by the heterologous expression of calcium channels containing ␣1A or ␣1B subunits (Sather et al, 1993; Grantham et al, 1994)
Summary
P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by -conotoxin MVIIC. 125I--Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by -conotoxins GVIA or MVIIA (Ki > 1 M), which are selective for N-type calcium channels. We have used 125IMVIIC to label calcium channels containing ␣1A subunits solubilized from rat cerebellar synaptosomes, and to explore their association with proteins of the secretory pathway. Our results indicate that a population of calcium channels containing ␣1A subunits can interact with a trimeric SNARE complex (Sollner et al, 1993a) composed of syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin).
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